Double electron–electron resonance (DEER) measures distances between spin labels attached at well-defined sites in a protein and thus has the potential to report on conformational states of proteins in cells. In this work, we evaluate the suitability of the small and rigid 4PS-PyMTA-Gd­(III) spin label for in-cell distance measurements. Three ubiquitin double mutants were labeled with 4PS-PyMTA-Gd­(III) and delivered into human HeLa cells by electroporation (EP) and hypotonic swelling (HS). Gd­(III)–Gd­(III) DEER measurements were carried out on cells frozen after different incubation times, following delivery to test the stability of the spin label inside the cell. For both delivery methods, it was possible to derive distance distributions up to 12 h after delivery, although we observed a decrease in the amount of the delivered protein with time. Surprisingly, only one mutant reported a significant change in the distance distribution with time and only for HS delivery. On the basis of in vitro exchange experiments with Mn­(II) and comparison with the same mutant labeled with BrPSPy-DO3MA-Gd­(III) and considering the presence of Mn­(II) in the cell, we hypothesized that the change occurred as a consequence of partial Gd­(III)/Mn­(II) exchange with endogenous Mn­(II). These experiments also showed that the relative Gd­(III)/Mn­(II) binding affinity depends on the labeling site in the protein, which accounts for the lack of change with the other mutants delivered under HS conditions. We conclude that 4PS-PyMTA-Gd­(III) is a good spin label for in-cell DEER for delivery by EP, but caution should be taken when HS is used.

The complexity of the cellular medium can affect proteins’ properties, and, therefore, in-cell characterization of proteins is essential. We explored the stability and conformation of the first baculoviral IAP repeat (BIR) domain of X chromosome-linked inhibitor of apoptosis (XIAP), BIR1, as a model for a homodimer protein in human HeLa cells. We employed double electron–electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd3+ spin labels at three representative protein residues, C12 (flexible region), E22C, and N28C (part of helical residues 26 to 31) in the N-terminal region. In contrast to predictions by excluded-volume crowding theory, the dimer–monomer dissociation constant KD was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was partially recapitulated under conditions of high salt concentrations, given that conserved salt bridges at the dimer interface are critically required for association. Unexpectedly, however, also the addition of the crowding agent Ficoll destabilized the dimer while the addition of bovine serum albumin (BSA) and lysozyme, often used to represent interaction with charged macromolecules, had no effect. Our results highlight the potential of DEER for in-cell study of proteins as well as the complexities of the effects of the cellular milieu on protein structures and stability.

GSH, Cys, Hcy, and H2S are important biothiols and play important roles in the living systems. Quantitative and simultaneous determination of these biothiols under physiological conditions is still a challenge. Herein, we developed an effective 19F-reactive tag that readily interacts with these four biothiols for the generation of stable thioether products that have distinguishable 19F-chemical shifts. These thioester compounds encode the characteristic fingerprint profiles of each biothiols, allowing one to simultaneously quantify and determine these biothiols by 1D 19F NMR spectroscopy. The intra-/extracellular GSH in live cells was assessed by the established strategy, and remarkable variations in the GSH stability were determined between the normal mammalian cells and cancer cells. It is notable that GSH hydrolyzes efficiently in the out-membrane of the cancer cells and the lysates. In contrast, GSH remains stable in the tested normal cells.